Tag Archives: hsp70

TDP43 and the anisosome

Neurologists have been interested in TDP43 (Tar Dna binding Protein of 43 kiloDaltons) for a long time. Mutants cause some cases of ALS (Amyotrophic Lateral Sclerosis — Lou Gehrig disease) and FTD (FrontoTemporal Dementia).  Some 50 different mutations in the protein have been found in cases of these two diseases.  Intracellular inclusions containing TDP are found in > 90% of sporadic ALS (no mutations) and 45% of FTD.

TDP43 contains 414 amino acids (as you might expect for a protein with a 43 kiloDalton mass).  There is an amino terminal ubiquitinlike fold, two RNA Recognition Motifs (RRMs) followed by a glycine rich low complexity sequence prion-like domain at the other (carboxy) end.  The disease causing mutations are found in the low complexity sequence. 

A  phase separated structure (the anisosome) never seen before involves  mutant TDP43 [ Science vol. 371 pp. 585, abb4309 pp. 1 –> 15 ’21 ].  It is a phase separated mass with liquid spherical shells and liquid cores.  The shells showed birefringence — evidence of a liquid crystal.  The cores show the HSP70 chaperone bound to TDP43 (which wasn’t binding RNA).

ATP is required to maintain the chaperone activity of HSP70. When ATP levels are reduced, the anisosome is converted into the protein aggregates seen in ALS and FTD.  So the anisosome is a protective mechanism. 

Biology is clearly leading chemistry around by the nose.  No chemist would ever have predicted something like this, or received a grant to mix all this stuff in a test tube not even thinking about stoichiometry and see what happened.  For more details on phase separation please see an old post — https://luysii.wordpress.com/2020/12/20/neuroscience-can-no-longer-ignore-phase-separation/

Here’s some stuff from that post to whet your appetite

Advances in cellular biology have largely come from chemistry.  Think DNA and protein structure, enzyme analysis.  However, cell biology is now beginning to return the favor and instruct chemistry by giving it new objects to study. Think phase transitions in the cell, liquid liquid phase separation, liquid droplets, and many other names (the field is in flux) as chemists begin to explore them.  Unlike most chemical objects, they are big, or they wouldn’t have been visible microscopically, so they contain many, many more molecules than chemists are used to dealing with.

These objects do not have any sort of definite stiochiometry and are made of RNA and the proteins which bind them (and sometimes DNA).  They go by any number of names (processing bodies, stress granules, nuclear speckles, Cajal bodies, Promyelocytic leukemia bodies, germline P granules.  Recent work has shown that DNA may be compacted similarly using the linker histone [ PNAS vol.  115 pp.11964 – 11969 ’18 ]

The objects are defined essentially by looking at them.  By golly they look like liquid drops, and they fuse and separate just like drops of water.  Once this is done they are analyzed chemically to see what’s in them.  I don’t think theory can predict them now, and they were never predicted a priori as far as I know.

No chemist in their right mind would have made them to study.  For one thing they contain tens to hundreds of different molecules.  Imagine trying to get a grant to see what would happen if you threw that many different RNAs and proteins together in varying concentrations.  Physicists have worked for years on phase transitions (but usually with a single molecule — think water).  So have chemists — think crystallization.

Proteins move in and out of these bodies in seconds.  Proteins found in them do have low complexity of amino acids (mostly made of only a few of the 20), and unlike enzymes, their sequences are intrinsically disordered, so forget the key and lock and induced fit concepts for enzymes.

Are they a new form of matter?  Is there any limit to how big they can be?  Are the pathologic precipitates of neurologic disease (neurofibrillary tangles, senile plaques, Lewy bodies) similar.  There certainly are plenty of distinct proteins in the senile plaque, but they don’t look like liquid droplets.

It’s a fascinating field to study.  Although made of organic molecules, there seems to be little for the organic chemist to say, since the interactions aren’t covalent.  Time for physical chemists and polymer chemists to step up to the plate.

 

Why drug discovery is so hard: Reason #27 Moonlighting effects.

Well, we all know what heat shock proteins (Hsps) do — they bind to proteins which have lost their shape due to heat (or other stressors), cuddle them hydrolyze ATP and nurse them back to health. But what  if some of them do other things? The phenomenon is called moonlighting.

The case of Hsp70 is instructive. Some background first. The Hsp70 chaperone transiently associates with its substrates in a manner controlled by its ATPase cycle. ATP binding to the amino terminal nucleotide binding domain (NBD) induces a conformational change in the carboxy terminal substrate binding domain (SBD) which results in low affinity for substrate. Hydrolysis of ATP converts the Hsp70 to the ADP state, which binds substrates with higher affinity. Exchange of ADP for ATP releases substrate completing the cycle. The hydrolysis of ATP is stimulated by J-domain containing cochaperones. These are the nucleotide exchange factors.  Back and forth Hsp70 and the damaged protein go through the cycle until the protein is nursed back to normal or, failing this, is destroyed.

The Hsp70 family acts early in protein synthesis by binding to a small stretch of hydrophobic amino acids on a protein’s surface. Aided by a set of smaller Hsp40 proteins (also known as J proteins), a hsp70 monomer binds to its target protein and then hydrolyzes ATP to ADP, undergoing a conformational change that causes the hsp70 to clamp down very tightly on the target. After the hsp40 dissociates (see below), the dissociation of the hsp70 protein is induced by the rapid rebinding of ATP after ADP release. Repeated cycles of hsp protein binding and release help the target protein to refold.

Enter [ Proc. Natl. Acad. Sci. vol. 112 pp. E3327 – E3336 ’15 ] This work shows Hsp70 is methylated on arginine #469 by Coactivator Associated aRginine Methyltransferase 1/Protein aRginine MethylTransferase 4 (CARM1/PRMT4) and demethylated by JuMonJi Domain containing 6 (JMJD6) — hideous acronyms shortening even more hideous names. Methylated Hsp70 then functions in transcription as a ‘regulator’ of Retinoid Acid Receptor beta 2 (RARbeta2) transcriptional acitivty. R468Mmethylated Hsp70 mediates the interaction between Hsp70 and TFIIH (Transcription Factor IIH).

The regulation of gene transcription is an entirely novel and unsuspected function for a heat shock protein. A classic example of moonlighting.

Drug chemists and pharmacologists are always concerned about off-target effects. For an interesting example please see https://luysii.wordpress.com/2011/02/02/medicinal-chemists-do-you-know-where-your-drug-is-and-what-it-is-doing/.  Off-target effects occur when their drug hits something else in the cell producing an unexpected (and usually untoward) effect.

If you are unaware that your target of choice is doing a little something else on the side (e.g. moonlighting) you can get an off target effect even when you hit your desired target. It’s a tough business. How many more moonlighters are out there that we don’t know about?

Hsp70 is a good example. Here are two more — no background provided, so you’re on your own — except to point out that glucocorticoids are a widely used class of drug.

[ Proc. Natl. Acad. Sci. vol. 112 pp. E1540 – 1549 ’15 ] Amazingly, the glucocorticoid receptor (GR)plays a role in mRNA degradation by acting as an RNA binding protein. When loaded onto the 5′ UnTranslated Region (5′ UTR) of a target mRNA, the GR recruits UPF1 through Proline-rich Nuclear Receptor Coregulatory protein 2 (PNRC2) in a ligand (of itself?) dependent manner to cuase rapid mRNA degradation. They call this GMD (Glurocorticoid receptor Mediated Decay). Along with Staufen Mediated mRNA Decay (SMD) and Nonsense Mediated mRNA Decay (NMD), they share UPF1 (Upstream Frameshift 1) and PNRC2.

[ Science vol. 323 pp. 723 – 724, 793 – 797 ’09 ] Stat3 proteins represent the canonical mediators of signals elicited by cytokines binding to type I cytokine receptors. However, GRIM19 (Gene associated with Retinoid Interferon Mortality 19), a mitochondrial protein, interacts with Stat3 and inhibits its transcriptional activity (where?). This work shows that Stat3 associates with GRIM19 containing complexes I and II (components of the electron transport chain) in mouse liver and muscle mitochondria. Levels of Stat3 in mitochondria are 10% of cytosolic levels.

Cells lacking Stat3 show decreased activity of mitochondrial complexes I and II. Effects on complex I and II don’t require Stat3’s DNA binding domain, the dimerization motif, or the tyrosine phosphorylation site controlling Stat3 nuclear localization and transcriptional activity — so this is a ‘moonlighting’ role for State3 having nothing to do with gene transcription. The serine phosporylation site on Stat3 is important. So Stat3 is required to maintain normal mitochondrial function.